CN113115853A - Preparation method of artificially-cultured second-generation giant salamander peptide freeze-dried powder and product thereof - Google Patents
Preparation method of artificially-cultured second-generation giant salamander peptide freeze-dried powder and product thereof Download PDFInfo
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- CN113115853A CN113115853A CN202110456272.2A CN202110456272A CN113115853A CN 113115853 A CN113115853 A CN 113115853A CN 202110456272 A CN202110456272 A CN 202110456272A CN 113115853 A CN113115853 A CN 113115853A
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- giant salamander
- dried powder
- freeze
- peptide
- molecular weight
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Links
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Marine Sciences & Fisheries (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to a preparation method of artificially cultured second-generation giant salamander peptide freeze-dried powder and a product, which comprises the following steps: (1) preparing artificially cultured second-generation giant salamander peptide freeze-dried powder. In the preparation process, the double-enzyme combination (papain and alkaline protease) enzymolysis ensures the thoroughness of enzymolysis, thereby ensuring the content of low molecular weight peptide segments of the product; meanwhile, ultra-low temperature crushing and sieving processes are combined, the molecular weight of the peptide is less than 500Da and is more than 70%, the whole process is carried out at low temperature, and the biological activity of artificially cultured second-generation giant salamander peptide is ensured. (2) Preparing artificial cultured second-generation giant salamander series products, including powder, tablets, oral liquid, other products and the like; (3) the product is taken for the first time by combining the sublingual administration technology and the oral mucosa absorption technology, and the product can be completely absorbed by the sublingual and the oral mucosa within 10s (seconds) by directly passing through the sublingual and the oral mucosa. After entering blood, the product can be placed in cells to exert the biological activity health care effect of the product.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a giant salamander peptide freeze-dried powder industrialized preparation technology taking giant salamander as a raw material and a series of giant salamander peptide freeze-dried powder products.
Background
Giant salamanders (Andrias davidianus) belong to the family of holotrichidae of the order uropila of the class amphibia. Is a national secondary protection animal and is listed in appendix I of International trade convention on endangered wild animal and plant species. The first national protection area-Hunan province Zhang kingdom giant salamander national level natural protection area in China was approved and established by the State Council in 1996. In 2011, giant salamanders in the kingdom of the family obtain national geographical sign protection products. The Pimpinella giant salamander resource protection and comprehensive utilization Hunan province engineering research center is established by combining Zhang Jiajiekang biotechnology limited company and Zhang Jiajie school district of Jichu university in 2015.
Giant salamanders have various applications in nutrition, health care, medicine and the like. (1) And (5) nutrition. The giant salamander is rich in protein, and the content of dry matter protein reaches 85 percent; the content of amino acid is high and can reach 91 percent, and especially the essential amino acid accounts for more than 40 percent of the total amount of the amino acid; the amino acid mode accords with a human body mode, the amino acid score of the giant salamander meat accords with a FAO score mode according to the FAO score mode and the content of essential amino acids of egg proteins serving as a score standard, and the total amount of the amino acid score of the giant salamander meat obviously exceeds the FAO score mode; the giant salamander is rich in various unsaturated fatty acids and trace elements, and particularly the content of selenium reaches 0.37mg/kg, so the giant salamander is also called 'life in water' and 'brain gold'. (2) And (5) health care. Giant salamanders are reported to have more than 100 active substances, such as active collagen peptide (collagen), epidermal active factor (CHF), oligo-glycopeptide, chondroitin sulfate, DHA, EPA, and the like. (3) And (5) the medicinal aspect. The record of the book Ben Cao Jing Ji Zhu (compendium of materia Medica) is: the giant salamander has the effects of improving intelligence, maintaining beauty and keeping young, enriching blood and promoting qi circulation, nourishing yin and tonifying kidney, preventing cancer, resisting cancer and the like, and has obvious curative effects on tonifying kidney and generating blood, treating anemia, blood channels and the like. Modern Chinese medicine holds that giant salamanders are sweet, flat and light in nature, and have the effects of tonifying qi, nourishing blood, benefiting intelligence, nourishing and strengthening. The giant salamander liver has the remarkable effects of improving eyesight, clearing heat and removing toxicity, dissolving heavy metal toxins, enriching blood and tonifying qi, and the giant salamander skin powder mixed with tung oil can treat burns and scalds, and particularly does not leave scars on the treatment of facial burns and scalds.
Development and application of giant salamander products. At present, development about giant salamander products covers a plurality of fields, including primary processed product, for example giant salamander chafing dish, giant salamander noodle, giant salamander snack food etc. and the product of intensive processing includes giant salamander albumen powder, giant salamander wine, giant salamander skin care products etc.. Generally, giant salamander products are mainly concentrated on primary processed products, (1) the cognition degree of the nation on the giant salamander is limited, so that the market of the processed products is not approved; (2) the research on giant salamanders is relatively lacked in domestic research institutes; (3) the main components of the giant salamander are in the muscle, oil, bone and viscera of the giant salamander. The content of main components of the giant salamander muscle is protein (the content of dry matter protein is more than 80 percent), the limited active components in the protein are peptide, but the giant salamander product processed by utilizing the giant salamander muscle is rough at present, which is shown in that on one hand, the molecular weight of the product is large, the peptide of the active component is not released, on the other hand, the processing technology has problems, the processed peptide has no biological activity, and the fishy smell of the product is heavy; meanwhile, the giant salamander protein powder products pass through digestive organs such as human oral cavity, stomach, small intestine and the like, and a plurality of active substances are decomposed, so that the products lose the original efficacy.
Sublingual administration techniques. Blood vessels under the tongue are abundant, and some medicines are placed under the tongue, so that the medicines can be directly absorbed into blood through the sublingual mucous membrane to exert curative effect, and the medicine is called as sublingual administration. Sublingual administration has the following characteristics: (1) the absorption is quicker. (2) The medicine is directly introduced into blood, so that the decomposition and the damage of an enzyme system in a digestive system to the medicine are avoided, the enzyme system cannot be metabolized by liver enzyme, the medicine effect is maintained, and the medicine is particularly significant for certain medicines. Especially has good effect on foods and medicines rich in biological activity.
At present, a plurality of patents for preparing giant salamander peptide by taking giant salamander as a raw material exist. Mainly comprises the following steps: (1) patent No. CN107446976A preparation method of giant salamander polypeptide powder, stopping feeding live giant salamander, slaughtering, cutting meat, removing fishy smell, baking, pulverizing, removing fat, performing enzymolysis, spray drying, etc. to obtain giant salamander peptide. (2) Patent No. CN110452949A "A method for preparing giant salamander meat polypeptide powder", comprises peeling giant salamander skin, cleaning giant salamander meat, inactivating, enzyme treating, centrifuging, and vacuum drying to obtain giant salamander meat polypeptide powder. Patent No. CN111363773A giant salamander active peptide, homogenizing giant salamander meat, adding synthase, centrifuging to obtain supernatant, separating with 3000Da ultrafiltration membrane separator, and freeze drying to obtain lyophilized powder. (3) Patent No. CN111172227A preparation method of a giant salamander small molecule polypeptide, giant salamander mucus, low-temperature vacuum drying, protease enzymolysis, enzyme deactivation, microporous membrane filtration, ultrafiltration of ultrafiltration membrane with cut-off molecular weight of 500 Dalton, and drying to obtain giant salamander small molecule polypeptide powder. (4) Patent No. CN103361393A preparation method of giant salamander oligopeptide, freeze drying giant salamander meat, pulverizing (below 60 mesh), extracting with edible oil extraction agent to remove fat, performing enzymolysis, filtering with dialysis membrane, inactivating enzyme, debitterizing, decolorizing, pasteurizing, concentrating, and freeze drying to obtain final product of giant salamander oligopeptide powder. (5) Patent No. CN105018555A preparation method of giant salamander skin collagen peptide, which comprises the steps of crushing giant salamander skin, removing fishy smell (sodium citrate and calcium chloride), stirring the giant salamander skin, performing ultrahigh pressure treatment, boiling the mixture, stirring and decoloring, centrifuging, performing microfiltration (the cut-off molecular weight is 4-5 kD) by using a filter membrane, freeze-drying and crushing to obtain the giant salamander skin collagen peptide.
In general, there is a commonality between the current research and patents on giant salamander peptides: firstly, raw materials: all adopt the relevant parts of the giant salamander as raw materials. And secondly, adopting an enzymolysis process, wherein most of the enzymolysis processes are single-enzyme enzymolysis processes. And thirdly, the method is only limited to experimental research. There are the following problems:
first, the industrialization cannot be realized. Is limited to experimental research and can not be produced in a pilot plant test and a large scale.
Secondly, the production process problem.
(1) The fishy smell of the final product is heavy due to the lack of a fishy smell removing process, and the product experience of customers is influenced.
(2) An enzymolysis process: the single enzyme enzymolysis is not thorough, resulting in lower content of small molecular peptide fragments.
(2) The process is difficult to industrialize. The enrichment of peptide is difficult to realize industrial production by adopting a microporous filter membrane for filtration.
(3) Drying processes, which result in the loss of biological activity of the peptide at high temperatures.
Thirdly, the main components of the giant salamander peptide product are unclear. The peptide content of that peptide fragment is unclear and the amino acid composition of the major peptide is unclear.
And fourthly, the giant salamander peptide product has no unified standard. There is no relevant data about the main physical and chemical indexes, the pollutant limit and the like.
And fifthly, the functional efficacy of the giant salamander peptide product is unclear, and the efficacy research of the giant salamander peptide product is not carried out.
In order to further make the giant salamander industry, ensure the industrialization of artificially cultured second-generation giant salamander peptide freeze-dried powder products and ensure the quality and the bioactivity of the products, the patent is drafted from the industrialization perspective, and the method comprises the steps of the giant salamander peptide industrialization process, the measurement of the content of the giant salamander peptide fragments, the standard preparation of the giant salamander peptide and the research on the effect of the giant salamander peptide products.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a preparation method of artificially cultured second-generation giant salamander peptide freeze-dried powder and a product thereof.
The technical scheme adopted by the invention is as follows: the preparation method of the artificially cultured second-generation giant salamander peptide freeze-dried powder comprises the following steps:
(1) killing a live giant salamander, removing mucus, skin, viscera and tail grease, and cleaning to obtain giant salamander muscle;
(2) mincing giant salamander muscle, and colloid milling;
(3) adjusting the temperature of the giant salamander after colloid milling to 50-55 ℃, adjusting the pH to 6-7, adding 1-5% of papain, carrying out enzymolysis for 1-3 hours, carrying out enzyme deactivation, adjusting the pH to 6-7, adding 5-8% of alkaline protease, carrying out enzymolysis for 1-2 hours, carrying out enzyme deactivation, and adjusting the pH to be neutral;
(4) freeze drying the enzymolysis liquid;
(5) carrying out low-temperature superfine grinding on the freeze-dried material;
(6) sieving, and sieving the superfine pulverized material with a 100-mesh sieve;
(7) sterilizing, and sterilizing the sieved material by 60Co irradiation with the irradiation dose of 3.0-5.0kGy to obtain the artificially cultured second-generation giant salamander peptide freeze-dried powder.
The peptide fragment content of the artificially cultured second-generation giant salamander peptide freeze-dried powder prepared by the preparation method is as follows: the molecular weight of the artificially cultured second-generation giant salamander peptide freeze-dried powder is more than 10000Da and accounts for 0% of the total weight by adopting high-efficiency size exclusion chromatography; the molecular weight of 5000-; the molecular weight of 3000-; 2000-3000Da accounts for 0.32% of the total amount; the molecular weight of 1000-2000Da accounts for 2.8 percent of the total weight; the molecular weight of 500-1000Da accounts for 19.8 percent of the total weight; the molecular weight <500Da was 77.05% of the total.
The molecular weight of the artificially cultured second-generation giant salamander peptide freeze-dried powder is 189-; the molecular weight <189Da represents 11.65% of the total.
The quality standard of the artificially cultured second-generation giant salamander peptide freeze-dried powder is as follows:
(1) physical and chemical properties and pollutant limits
The protein is more than or equal to 75 (g/100 g), the peptide content is more than or equal to 90 (g/100 g), the proportion of protein hydrolysate with the relative molecular mass of less than 500Da is more than or equal to 70 (g/100 g), the ash content is less than or equal to 10 (g/100 g), the water content is less than or equal to 10 (g/100 g), the crude fat (calculated by dry basis) is less than or equal to 0.5 (g/100 g), the inorganic arsenic (As) is less than or equal to 0.5 (mg/kg), the lead (Pb) is less than or equal to 0.5 (mg/kg), the cadmium (Cd) is less than or equal to 0.1 (mg/kg), and the methyl mercury (Hg) is less than or equal to 0.5;
(2) nutrient composition
1689 kilojoules (kJ) of energy, 76.4 grams (g) of protein, 8.7 grams (g) of fat, 4.0 grams (g) of carbohydrate, 823 milligrams (mg) of sodium, 37 micrograms (g) of selenium.
And preparing the artificially cultured second-generation giant salamander peptide freeze-dried powder into powder, oral liquid or tablets.
The giant salamander peptide freeze-dried powder, oral liquid or tablets can be completely absorbed through sublingual and oral mucosa within 10 s.
The invention has the beneficial effects that:
(1) the process flow can be industrialized: cleaning, mincing, protease enzymolysis, freeze drying, crushing, sieving, sterilization and packaging, and each process can be produced in a large scale and is not limited to experimental research.
(2) The quality of the product is ensured by double-enzyme combined enzymolysis: the enzymolysis thoroughness is ensured by papain and alkaline protease, the content of the molecular weight peptide section of the product is further ensured, and the molecular weight of the peptide is ensured to be more than 70% in a ratio of less than 500Da by combining the ultra-low temperature crushing and sieving processes.
(3) The low temperature process ensures the biological activity: the whole process is carried out at low temperature, so that the spatial structure of artificially cultured second-generation giant salamander peptides is ensured, and the biological activity of the product is further ensured.
(4) The giant salamander product combines a sublingual administration technology and an oral mucosa absorption technology for the first time. Macromolecular energy substances, proteins, fats and the like can only be digested and absorbed through oral cavity, stomach, intestinal tract and the like, but cannot pass through sublingual and oral mucosa; meanwhile, the penetration capacity of the product is closely related to the lipid solubility, the molecular weight and the like of the product, and the effective absorption penetration enhancer can be selected to promote the product to penetrate and absorb into sublingual blood. The giant salamander muscle enzymolysis is carried out to obtain small molecular peptides (the molecular weight is less than 500Da and accounts for more than 70%) to ensure that the product can effectively pass through sublingual and oral mucosa, and meanwhile, the product can effectively pass through sublingual and oral mucosa barriers and enter blood to directly play a role in a human body by selecting and absorbing the penetration enhancer.
Detailed Description
Firstly, preparing artificially cultured second-generation giant salamander peptide freeze-dried powder.
1. And (4) preparing raw materials.
Killing a living giant salamander, scraping off mucus on the body surface, peeling, removing internal organs, removing tail grease, and cleaning to obtain giant salamander muscle.
2. And (4) preparing a colloid-milled giant salamander liquid.
Giant salamander muscle dicing, mincing by a mincing machine, colloid milling and colloid milling, and colloid milling of giant salamander liquid.
3. And (4) enzymolysis.
Colloid milling giant salamander liquid, adjusting the temperature to 50 ℃, adjusting the pH value to 6.5, adding 3% of papain, carrying out enzymolysis for 1.5 hours, carrying out enzyme deactivation, adjusting the pH value to 6.5 again, adding 6% of alkaline protease, carrying out enzymolysis for 2 hours, carrying out enzyme deactivation, and adjusting the pH value to be neutral.
4. And (5) freezing and drying the enzymolysis liquid.
Pre-freezing: the freezing temperature is-30 ℃ and the time is 2.5 hours; vacuum freeze drying: the sublimation drying temperature is-22 ℃, the freezing pressure is 15Pa, and the drying time is 10 hours; the desorption temperature was 10 ℃ and the pressure was 180Pa, and the time was 3.5 hours.
5. And (3) carrying out low-temperature superfine grinding on the freeze-dried material.
6. Sieving, and sieving with 300 mesh sieve.
7. Sterilizing, and sterilizing the sieved material by 60Co irradiation with the irradiation dose of 3.0-5.0kGy to obtain the artificially cultured second-generation giant salamander peptide freeze-dried powder.
And secondly, measuring the content of the peptide fragment of the giant salamander.
1 assay method-high performance size exclusion chromatography is used for assay.
Separating according to the difference of molecular volume of sample components by using porous filler as a stationary phase, detecting under the condition of ultraviolet absorption wavelength of peptide bond of 220nm, processing chromatograms and data of a standard product and a sample by using special data processing software (namely GPC software) for measuring the relative molecular mass distribution, and calculating the relative molecular mass size and the distribution range of the protein peptide according to a relative molecular mass correction curve equation.
2, reagent.
(1) Acetonitrile and chromatographic purity. Trifluoroacetic acid and analytically pure. Ultrapure or redistilled water.
(2) Standard substance used for relative molecular mass calibration curve
Cytochrome C (MW 12384), aprotinin (MW 6512), bacitracin (MW 1423), ethionin-tyrosine-arginine (MW 451), and ethionin-ethionin (MW 189).
3 experimental parameters.
TSKgel G2000SWXL300mm X7.8 mm (GEL LOT 502R) or other GEL columns of the same type having properties similar thereto which are suitable for determining the molecular weight distribution of peptides.
The volume ratio of the mobile phase, the acetonitrile, the water and the trifluoroacetic acid is 40: 60: 0.05.
Detection wavelength: UV220 nm. The flow rate is 0.5-0.8 mL/min.
The column temperature is 30-35 ℃. The injection volume is 10 mu L-15 mu L.
The chromatographic system meets the detection requirement, and under the chromatographic condition, the column effect of the gel chromatographic column, namely the theoretical plate number N is not less than 5000 calculated according to the peak of a tripeptide standard product (alanine-alanine), and the distribution coefficient Ka of the protein peptide is between 0 and 1.
4. And (5) preparing a relative molecular mass calibration curve.
Respectively preparing the peptide standard substance solutions with different relative molecular masses by using mobile phase matching, filtering by using a polytetrafluoroethylene or nylon filter membrane with the aperture of 0.2-0.5 mu m, and then respectively injecting samples to obtain the chromatograms of the series of standard substances. The relative molecular mass calibration curve and its equation are obtained by plotting the logarithm of the relative molecular mass (lgMW) against the retention time or by linear regression.
5. And (4) preparing a sample.
Weighing about 125.0 mg of a sample in a 25 mL volumetric flask, fixing the volume to a scale with a mobile phase, ultrasonically oscillating for 10min to fully dissolve and uniformly mix the sample, filtering by using a polytetrafluoroethylene or nylon filter membrane with the aperture of 0.2-0.5 mu m, and using the filtrate for determination.
6. Calculation of relative molecular mass.
And (3) carrying out sample injection analysis on the prepared sample solution under the A.4 chromatographic condition, and then using GPC data processing software to calculate and process the chromatogram and the data of the sample according to a relative molecular mass calibration curve equation, so as to obtain the relative molecular mass size and the distribution range of the protein peptide in the sample. The sum of the relative percentages of protein hydrolysates with relative molecular masses below 5000 was calculated by peak area normalization.
7. And (6) detecting the result.
Remarking: ND = not detected.
Thirdly, artificially breeding the indexes of the second-generation giant salamander peptide product.
The main indexes of the product are determined according to the regulations of the food safety law of the people's republic of China, the Standard Law of the people's republic of China.
1. Physical and chemical indexes and pollutant limit.
2. The product contains nutrient components.
Fourthly, measuring sublingual permeability, absorption and permeation enhancer selection and permeation time of the giant salamander peptide freeze-dried peptide.
1. And (3) measuring the sublingual permeability of the giant salamander peptide freeze-dried powder.
The permeability is closely related to the lipid solubility, molecular weight and the like of the product. Generally, polypeptide drugs with molecular weight less than 300Da can be absorbed sublingually. The influence of the molecular weight of the giant salamander peptide freeze-dried powder on the sublingual permeability is determined.
(1) And (3) preparing peptide fragments of different giant salamander peptide freeze-dried powders.
A. The giant salamander enzymolysis liquid is prepared by the process;
B. adopting a device with the molecular weight cut-off less than 1000Da, the molecular weight cut-off less than 700Da, the molecular weight cut-off less than 500Da and the molecular weight cut-off less than 300 Da.
C. Collecting the enzymolysis liquid passing through the molecular weight cut-off, and placing the enzymolysis liquid in a freeze-drying device for freeze-drying to obtain the giant salamander peptide freeze-dried powder peptide segment.
(2) And (3) measuring the penetration and absorption capacities of different giant salamander peptide freeze-dried powder peptide fragments.
A. Selecting 40 people of different ages, male and female, and performing sublingual oral measurement
B. And (6) measuring results.
Different groups of people and different peptide fragment absorption conditions
Complete absorption time of different peptide fragments of different populations
2. Giant salamander peptide freeze-dried powder absorption and permeation enhancer selection
More than 90% of the giant salamander freeze-dried powder produced by the giant salamander peptide freeze-dried powder production process is the giant salamander peptide freeze-dried powder with the molecular weight of less than or equal to 1000Da and the molecular weight of less than 500Da, and the giant salamander peptide freeze-dried powder can be absorbed. Therefore, the osmotic absorbent is selected for the giant salamander peptide freeze-dried powder with the molecular weight of less than 1000 Da.
The absorption penetrant is selected from propylene glycol, ethanol, and beta-cyclodextrin which can be directly eaten by human body, and is added according to the dosage of 0.1%, 0.5% and 1%.
The sublingual oral test is carried out by selecting 40 people of different age, male and female in different stages.
Influence of propylene glycol on absorption condition of giant salamander peptide freeze-dried powder
Influence of ethanol on absorption condition of giant salamander peptide freeze-dried powder
Influence of beta-cyclodextrin on absorption condition of giant salamander peptide freeze-dried powder
And fifthly, preparing products of different formulations of the giant salamander peptide freeze-dried powder.
1. The giant salamander peptide freeze-dried powder is directly used as a finished product.
A. Giant salamander peptide freeze-dried powder: slaughtering (live giant salamander slaughtering, mucus removal, skin removal, viscera removal and tail oil removal) — colloid milling (giant salamander muscle mincing) — enzymolysis (adjusting the temperature to 50-55 ℃, pH6-7, adding 1% -5% of papain, enzymolysis for 1-3 hours, enzyme inactivation, further adjusting pH6-7, adding 5% -8% of alkaline protease, enzymolysis for 1-2 hours, enzyme inactivation, and pH adjustment to neutrality) — freeze drying (enzymolysis liquid freeze drying) ultra-low temperature pulverization (freeze-dried material is subjected to ultra-micro pulverization), sieving (the material subjected to ultra-micro pulverization is sieved by a 100-mesh sieve) — sterilization (the sieved material is subjected to irradiation sterilization by 60Co, and the irradiation dose is 3.0-5.0 kGy), and) -artificial breeding of second-generation giant salamander peptide freeze-dried powder.
B. The giant salamander peptide freeze-dried powder product form is as follows: powder (including peptide powder, peptide freeze-dried powder and solid beverage), oral liquid (including beverage and oral liquid), tablet (including tablet candy and tablet), and other products (prepared from giant salamander peptide freeze-dried powder as raw material).
2. Other products using giant salamander peptide freeze-dried powder as raw material
Powder preparation: giant salamander peptide freeze-dried powder raw material, packaging and sterilizing.
Oral liquid: giant salamander peptide freeze-dried powder raw material-batching-filling-sterilization.
And (3) tablet preparation: giant salamander peptide freeze-dried powder raw material-batching-mixing-granulating-drying-granulating-tabletting-coating.
And others: other products produced by taking giant salamander peptide freeze-drying as a raw material.
Claims (6)
1. The preparation method of the artificially cultured second-generation giant salamander peptide freeze-dried powder is characterized by comprising the following steps of:
(1) killing a live giant salamander, removing mucus, skin, viscera and tail grease, and cleaning to obtain giant salamander muscle;
(2) mincing giant salamander muscle, and colloid milling;
(3) adjusting the temperature of the giant salamander after colloid milling to 50-55 ℃, adjusting the pH to 6-7, adding 1-5% of papain, carrying out enzymolysis for 1-3 hours, carrying out enzyme deactivation, adjusting the pH to 6-7, adding 5-8% of alkaline protease, carrying out enzymolysis for 1-2 hours, carrying out enzyme deactivation, and adjusting the pH to be neutral;
(4) freeze drying the enzymolysis liquid;
(5) carrying out low-temperature superfine grinding on the freeze-dried material;
(6) sieving, and sieving the superfine pulverized material with a 100-mesh sieve;
(7) sterilizing and sievingThe latter material is passed through60Co irradiation is used for sterilization, and the irradiation dose is 3.0-5.0kGy, so that the artificially cultured second-generation giant salamander peptide freeze-dried powder is obtained.
2. The artificially cultured secondary giant salamander peptide freeze-dried powder prepared by the preparation method according to claim 1, wherein the content of peptide fragments of the artificially cultured secondary giant salamander peptide freeze-dried powder is as follows: the molecular weight of the artificially cultured second-generation giant salamander peptide freeze-dried powder is more than 10000Da and accounts for 0% of the total weight by adopting high-efficiency size exclusion chromatography; the molecular weight of 5000-; the molecular weight of 3000-; 2000-3000Da accounts for 0.32% of the total amount; the molecular weight of 1000-2000Da accounts for 2.8 percent of the total weight; the molecular weight of 500-1000Da accounts for 19.8 percent of the total weight; the molecular weight <500Da was 77.05% of the total.
3. The artificially cultured secondary giant salamander peptide freeze-dried powder prepared by the preparation method according to claim 2, wherein the molecular weight of the artificially cultured secondary giant salamander peptide freeze-dried powder is 189-500Da in the total amount of 65.4%; the molecular weight <189Da represents 11.65% of the total.
4. The artificially cultured secondary giant salamander peptide freeze-dried powder prepared by the preparation method according to claim 1, wherein the quality standard of the artificially cultured secondary giant salamander peptide freeze-dried powder is as follows:
(1) physical and chemical properties and pollutant limits
The protein is more than or equal to 75 (g/100 g), the peptide content is more than or equal to 90 (g/100 g), the proportion of protein hydrolysate with the relative molecular mass of less than 500Da is more than or equal to 70 (g/100 g), the ash content is less than or equal to 10 (g/100 g), the water content is less than or equal to 10 (g/100 g), the crude fat (calculated by dry basis) is less than or equal to 0.5 (g/100 g), the inorganic arsenic (As) is less than or equal to 0.5 (mg/kg), the lead (Pb) is less than or equal to 0.5 (mg/kg), the cadmium (Cd) is less than or equal to 0.1 (mg/kg), and the methyl mercury (Hg) is less than or equal to 0.5;
(2) nutrient composition
1689 kilojoules (kJ) of energy, 76.4 grams (g) of protein, 8.7 grams (g) of fat, 4.0 grams (g) of carbohydrate, 823 milligrams (mg) of sodium, 37 micrograms (g) of selenium.
5. The artificially cultured secondary giant salamander peptide freeze-dried powder prepared by the preparation method according to claim 1, wherein the artificially cultured secondary giant salamander peptide freeze-dried powder is prepared into powder, oral liquid or tablets.
6. The artificially cultured secondary giant salamander peptide freeze-dried powder prepared by the preparation method of claim 1, wherein the powder, oral liquid or tablet of the giant salamander peptide freeze-dried powder can be completely absorbed by sublingual and oral mucosa for 10 s.
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